The Construction Of High-pathogeniticity Island Deletion Mutants Of K88ac+ Enterotoxigenic Escherichia Coli And Related Virulence Function Analysis

Enterotoxigenic Escherichia coli(ETEC)is the most common pathogenic E.coli which can cause neonatal and post-weaning piglets diarrhea,and result in high morbidity and mortality in swine production.The high-pathogeniticity island(HPI)was first identified in high-pathogeniticity Yersinia,which relates to the synthesis,uptake and transport of siderophore and associated with the virulence and pathogenicity of Yersinia and mouse virulence.In this study,we designed following assays to explore the function of HPI in ETEC C83902.1.The construction of HPI knock-out mutants via CRISPR/Cas9Based on the whole genome sequencing results of C83902 from our laboratory,we designed primers for HPI deletion which can fuse the sgRNA gene and the upstream and downstream homologous gene of the HPI to the plasmid pTarget to construct a recombinant plasmid pTargetT,which can transcript sgRNA and provide donor DNA.We electroprated the plasmid pTargetT and pCas which containing the gene that can translate both the Cas9 protein and λ-RED recombination system into C83902,to knockout the HPI without any mark.The constructed C83902△HPI mutant was further confirmed by PCR amplication.2.The function of HPI in pathogenicity of K88ac+ enterotoxigenic Escherichia coliWith the constructed C83902△HPI mutant,we compared and analyzed related biological characteristics of C83902 and the mutant.In vitro growth test shows that under the same culture condition,the growth rate of wild type C83902 and the mutant was basically the same at all stages of the growth cycle.Likewise,the CAS assay shows that there were no significant difference in siderophore synthesis between C83902 and the mutant.However,the biofilm-formation ability of C83902△HPI reduced 50.97%(P<0.05)and the adhesion ability of the mutant to IPEC-J2 cells reduced 23.3%.Furthermore,Real-Time PCR was performed to detect the important virulence factors on the transcription level,such as K88 fimbriae,sfm fimbriae,serine protease,type I fimbriae,E.coli common pili,capsule,alpha fimbriae,Salmonella typhimurium fimbriae,flagella,hemolysin and curli fimbriae.The results showed that compared to WT strain,C83902△HPI reduced fae G by 12.02%,sfmH by 15.58%,sepA by 37%(P<0.01),fimH by 11.78%,ecpA by 30.4%(P<0.05),kpsDby 37.5%(P<0.05),cblB by 37.75%(P<0.05),stfG by 34,84%(P<0.01),ipfA by 35.3%(P<0.01),fliC by 41.2%(P<0.01)on the transcription level.Above all,we concluded that HPI in ETEC affects the biofilm formation and regulates the expression of sepA,ecpA,kpsD,cblB,stfG,ipfA.fliC and other related genes,thus affects virulence.But HPI is not the only gene that can synthesis the siderophore in ETEC C83902.